1 Environmental Microbiology Achimer October 2015, Volume 17, Issue 10, Pages 4035-4049 http://dx.doi.org/10.1111/1462-2920.12955 http://archimer.ifremer.fr http://archimer.ifremer.fr/doc/00275/38594/ © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing Massana Ramon 1 * , Gobet Angélique 2, 3, Audic Stéphane 2, 3, Bass David 4, 5, Bittner Lucie 2, 3, 6, Boutte Christophe 2, 3, Chambouvet Aurélie 7, Christen Richard 8, Claverie Jean-Michel 9, Decelle Johan 2, 3, Dolan John R. 10, Dunthorn Micah 6, Edvardsen Bente 11, Forn Irene 1, Forster Dominik 6, Guillou Laure 2, 3, Jaillon Olivier 12, Kooistra Wiebe H. C. F. 13, Logares Ramiro 1, Mahé Florent 6, Not Fabrice 2, 3, Ogata Hiroyuki 14, Pawlowski Jan 15, Pernice Massimo C. 1, Probert Ian 2, 3, Romac Sarah 2, 3, Richards Thomas 7, Santini Sébastien 9, Shalchian-Tabrizi Kamran 11, Siano Raffaele 16, Simon Nathalie 2, 3, Stoeck Thorsten 6, Vaulot Daniel 2, 3, Zingone Adriana 13, De Vargas Colomban 2, 3 1 Institut de Ciències del Mar (CSIC); ES-08003 Barcelona Catalonia ,Spain 2 Ecologie Systematique Evolution; CNRS; FR-29682 Roscoff, France 3 UMR7144 - Equipe EPPO: Evolution du Plancton et PaléoOcéans; UPMC Université Paris 06; Roscoff France 4 The Natural History Museum; London SW7 5BD, UK 5 Cefas; Weymouth Dorset DT4 8UB, UK 6 University of Kaiserslautern; D-67663 Kaiserslautern ,Germany 7 Biosciences; University of Exeter; Exeter EX4 4QD ,UK 8 CNRS; UMR 7138; Université Nice Sophia Antipolis; FR-06108 Nice ,France 9 CNRS; UMR 7256; Aix-Marseille Université; FR-13288 Marseille ,France 10 CNRS; UMR 7093; UPMC Université Paris 06, Laboratoire d'Océanographie de Villefranche; FR- 06230 Villefranche-sur-Mer France 11 Department Biosciences; University of Oslo; N-0316 Oslo ,Norway 12 CEA; Genoscope, 2 rue Gaston Crémieux; FR-91000 Evry, France 13 Stazione Zoologica Anton Dohrn; Villa Comunale; I-80121 Naples, Italy 14 Institute for Chemical Research, Kyoto University; Uji Kyoto 611-0011, Japan 15 University of Geneva, CH-1211 Geneva ,Switzerland 16 Ifremer, DYNECO/Pelagos; BP 7029280 Plouzané, France * Corresponding author : Ramon Massana, Tel: (+34) 93 2309500; Fax: (+34) 93 2309555 ; email address : [email protected] Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. 2 Abstract : Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date. Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. Page 3 of 38 Introduction Protists or unicellular eukaryotes cover a wide spectrum of cell sizes, shapes, and taxonomic 60 affiliations (Schaechter, 2012). They represent the majority of eukaryotic lineages, so studying their diversity is of primary interest for understanding the eukaryotic tree of life (Keeling etFor al., 2005; Burki,Peer 2014). Moreover, Review protists play a variety Only of crucial roles in marine ecosystems from primary producers, predators, decomposers to parasites (Sherr et al., 2007), leading to much effort in quantifying particular species and inferring their ecological 65 functions. A vast literature exists in which species of dinoflagellates (e.g. Graham et al., 2004), diatoms (e.g. Olguín et al., 2006) and ciliates (e.g. Dolan et al., 2013) have been studied based on morphological features observable in light microscopy (LM), a task that requires considerable expertise and time to key out species accurately. Even for these relatively visible groups, examples are known of morphologically similar individuals 70 belonging to different cryptic species (Amato et al., 2007) or morphologically distinct types from the same species (Pizay et al., 2009). Accurate identification may thus not always be discerned from cell morphology alone, and this is more critical for protists below 20 µm in size that often lack conspicuous shapes (Massana, 2011). Over the last decades, DNA sequencing of environmental phylogenetic markers has changed our perception of microbial 75 diversity in most ecosystems. These molecular surveys have been instrumental in decoding the large protist diversity and in unveiling new lineages, such as Picozoa (Not et al., 2007; Seenivasan et al., 2013), MALV (Marine Alveolates) clades (Guillou et al., 2008) and MAST (Marine Stramenopiles) clades (Massana et al., 2004; 2014). Earlier molecular surveys were based on clone libraries of near full-length 18S rDNA genes 80 followed by Sanger sequencing of a subset of the clones (Díez et al., 2001; López-García et al., 2001; Moon-van der Staay et al., 2001). The resulting high-quality, often manually Wiley-Blackwell and Society3 for Applied Microbiology Page 4 of 38 checked environmental sequences have been crucial for the phylogenetic placement of novel clades and, together with sequences from monoclonal cultures, are the basis of reference rDNA databases (Guillou et al., 2013; Pernice et al., 2013). However, traditional clone 85 libraries only capture the most dominant species in the community (Pedrós-Alió, 2006), a limitation bypassed by high-throughput sequencing (HTS) methods. By providing the deep inventoriesFor needed both Peer for taxonomic descriptionsReview and sample comparisons,Only HTS has enabled microbial ecology to advance greatly. HTS has been applied to study protist diversity in a wide variety of systems, including surface and deep marine waters (Amaral-Zettler et al., 90 2009; Cheung et al., 2010; Edgcomb et al., 2011; de Vargas et al., 2015), marine sediments (Bik et al., 2012), lakes (Mangot et al., 2013), soils (Bates et al., 2013), and metazoan hosts (He et al., 2014). In the case of marine protists, most studies have targeted a specific size- fraction or a particular location. In addition, these surveys generally used environmental DNA as template for PCR amplification, and it has been shown that using RNA extracts instead can 95 provide a different picture of biodiversity (Stoeck et al., 2007; Not et al., 2009; Lejzerowicz et al., 2013) and useful complementary information (Blazewicz et al., 2013). The present study is an investigation of benthic-pelagic protists in marine habitats along the European coastline, sampled between 2009 and 2010 during the research program BioMarKs. The 95 different pyrosequenced samples analyzed herein address total protist diversity from 100 benthic and planktonic (size-fractionated) communities using an eukaryotic "universal" primer set to PCR amplify the V4 rDNA pre-barcode (Pawlowski et al., 2012) from both DNA and RNA extracts. Previous studies using this sequencing dataset focused on particular taxonomic groups, such as uncultured MAST (Logares et al., 2012), cercozoan amoebae (Berney et al., 2013) or diatoms (Nanjappa et al., 2014). More recently, we used a subset of 105 the samples (23 planktonic RNA samples) and newly collected HTS reads (Illumina sequencing of the V9 18S rDNA region) to investigate the patterns of a particular community Wiley-Blackwell and Society4 for Applied Microbiology Page 5 of 38 property, the rare biosphere (Logares et al., 2014). Here we analyze the complete 454 dataset from a taxonomic community perspective to address the following questions: How different are the protist communities found in the pico-, nano-, micro/mesoplankton and sediments? 110 Does the dissolved DNA fraction originate from particular taxonomic groups and/or organismal size-fractions? Do DNA and RNA surveys provide similar protist diversity profiles? WhichFor taxonomic Peer groups are differentiallyReview represented Only in either survey? Which groups dominate in each plankton organismal size fraction and associated sediments? Overall, our study highlights fundamental questions on the diversity of protists, an important but less 115 known component of marine